The long-term objectives of this project are to define how intracellular Ca/2+ transport processes are altered by hippocampal neurons during aging and Alzheimer's disease, and to determine the role these changes in Ca/2+ homeostasis have in neurodegeneration. The project's immediate goal is to test the specific hypothesis that a common feature of both neuronal aging and the toxicity of beta-amyloid peptide is that they increase calcium-related stress on mitochondria in hippocampal neurons. The research project will address the following specific aims in order to test various features of the central hypothesis. 1) Test the hypothesis that beta-amyloid toxicity alters the functional balance between various Ca/2+ extrusion and sequestration mechanisms so as to increase the Ca/2+ load mitochondria Ca/2+ transport, increasing the time-averaged exposure of mitochondria to elevations of [Ca/2+]. 3) Evaluate the hypothesis that the likelihood that cytosolic Ca/2+ ions get taken up into mitochondria depends on the route with which the Ca/2+ ions enter the cytosol. 4) Test the hypothesis that a) aging enhances the ability of Ca/2+ ions to induce reactive oxygen species (ROS) production in hippocampal mitochondria, and b) that such Ca/2+-induced production of ROS makes an important contribution of the overall oxidative stress induces by beta-amyloid peptides. 5) Evaluate the hypothesis that aging and beta-amyloid peptides sensitize the mitochondrial permeability transition pore to intramitochondrial Ca/2+ ions. These aims will be investigated using two experimental models. Beta- amyloid toxicity will be studied in rat hippocampal neurons in primary culture, while the effects of aging will be studied in rat hippocampal "zipper" slices. Ca/2+ homeostasis will primary be studied in situ, using a combination of pharmacological, electrophysiological, microfluorimetric, and cell imaging methods. A particular strength of this project is the use of advanced confocal imaging methods to evaluate mitochondrial [Ca/2+] and membrane potentials in situ.